Project Summary/Abstract Circulating tumor cells (CTCs) are cancer cells that are detached from primary solid tumors and carried through the vasculature to potentially seed distant site metastases in vital organs ? the main cause of death by cancer. Single-cell molecular characterizations and analyses of CTCs not only could benefit basic cancer research, but also might eventually lead to a more effective cancer treatment. However, one major limitation of CTCs in cancer research and its clinical applications has been the limited availability of viable CTCs for investigations, due in part to the small patient blood volumes that are allowable for research, which usually yielded typical less than 1-10 CTCs from 1 mL of whole blood. Current devices, including both label-based and label-free ones had their limitations. Our goal is to develop a microfluidic technology that combines both label-based and label-free methods to enrich CTCs based on size and biomarkers to achieve high purity and high recovery rate simultaneously. The objective of this application is to develop an integrated ferrohydrodynamic cell separation (iFCS) method that is tumor cell antigen-independent and cell size size-inclusive, and allows for depletion of WBCs and enrichment of CTCs regardless of tumor antigen and cell sizes, resulting in comprehensive recovery of intact CTCs with minimal WBC contamination that are suitable for phenotypic analysis. Aim 1: We aim to further develop integrated ferrohydrodynamic cell separation (iFCS) method for tumor antigen independent and cell size inclusive enrichment of clinical CTCs, with a goal of enriching the entire repertoire of intact CTCs with minimal WBC contamination. Aim 2: We aim to validate iFCS with 36 breast cancer patient samples for its goal of enriching the entire repertoire of intact CTCs with minimal WBC contamination